Due to the unusual functional hemizygosity of the resident genes, imprinted regions have been linked to several different kinds of human genetic defects, ranging from neurological defects to cancer. We have characterized one imprinted domain located in human chromosome 19q13.4 (HSA19q13.4), and this interval is also associated with imprinting related genetic disorders in human and mouse. This domain contains at least 6 imprinted genes, including Peg3, Usp29, Zim3/Usp29-as, Zim1, Zim2, and Zfp264. As in other imprinted domains gene clustering is thought to reflect the presence of long-range controlling mechanism(s). The long-term goal of this work is to understand how the imprinted expression of each gene within this domain is regulated;the proposed project is focused specifically on the regulation of two paternally expressed conserved genes, Peg3 and Usp29. Comparative genomic studies allowed us to identify one differentially methylated region that we believe may serve as an Imprinting Control Region (ICR) for this interval. This potential ICR overlaps with the promoter region of Peg3 and Usp29, termed P1-DMR (Promoter 1-Differentially Methylated Region). We have also identified an evolutionarily conserved Gli-type zinc-finger gene YY1 as a methylation-sensitive trans factor for P1-DMR. We predict that P1-DMR/YY1 may function either as a methylation sensitive chromosomal insulator or neural enhancer for the imprinting control of the neighboring genes. To investigate the potential function of P1-DMR for imprinting control, we will analyze the insulator and promoter activity observed from P1-DMR using several cell-line based assay systems. Mutant mice carrying targeted deletions of P1-DMR will be generated to test the in vivo function of P1-DMR, and the potential roles of YY1 in the P1-DMR function will also be investigated using transgenic mice inheriting various reporter constructs. These results will provide new insights into the regulation of this imprinted region, and explore the possible role of YY1 as a trans factor for mammalian genomic imprinting. These studies will provide new clues to imprinting-related phenotypes linked to this domain in human and mouse.